Nitrogen Affects Anthocyanin Biosynthesis by Regulating MdLOB52 Downstream of MdARF19 in Callus Cultures of Red-Fleshed Apple (Malus sieversii f. niedzwetzkyana)

摘要

Nitrogen limitation stress is known to markedly promote anthocyanin accumulation and upregulate anthocyanin biosynthesis-related genes; however, the underlying molecular mechanisms have not been fully characterized. In this study, a callus was induced from the leaves of an R6 homozygous red-fleshed apple, the hybrid offspring of Malus sieversii f. niedzwetzkyana and Malus x domestica 'Fuji.' We analyzed the growth and accumulation of anthocyanin in callus under different nitrogen concentrations (0.01, 0.02, 0.04, and 0.06 M NO3 (-)). The results showed that the callus growth in the 0.01 M NO3 (-) condition was minimal, but its anthocyanin content was significantly elevated compared with those under the other nitrogen treatments. As the concentration of nitrogen increased from 0.01 to 0.04 M, the expression of the anthocyanin structural genes, MdCHS and MdDFR, and the transcription factor genes MdMYB9, MdMYB10, MdbHLH3, and MdbHLH33 were downregulated. On the contrary, the transcript levels of MdLOB52 and MdARF19 were increased by the nitrogen treatments. The overexpression of MdLOB52 inhibited anthocyanin accumulation in the callus. Subsequent yeast one-hybrid assays showed that MdARF19 can directly bind the promoter of MdLOB52, which may strengthen the activation of MdLOB52 and affect anthocyanin accumulation. Our findings provide new insights into the mechanisms by which the LOB genes respond to nitrogen to regulate anthocyanin biosynthesis in a red-fleshed apple callus.